PROJECT SUMMARY/ABSTRACT This application is responsive to PA-17-440: The Interplay of Cell Death Pathways in Cancer Cell Survival and Resistance to Therapy (R01). Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the US. Most CRC patients are not responsive to therapeutic treatment. Induction of programmed cell death, widely known as apoptosis, is a key effect of anticancer therapy. Recent studies indicate that programmed cell death is not confined to caspase-dependent apoptosis, but includes necroptosis, a regulated form of necrotic death controlled by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like protein (MLKL). Accumulating evidence suggests that necroptosis functions as a defensive mechanism against oncogenic mutations and pathogens, and can be utilized by a variety of anticancer agents to kill cancer cells. However, the regulatory mechanisms and functional role of necroptosis in anticancer therapy are poorly understood. Despite extensive efforts for restoring apoptosis in cancer cells, few attempts have been made to manipulate necroptosis for improving anticancer therapy, largely due to insufficient understanding of this newly defined cell death modality. Our preliminary data show that frequent loss of RIP3 expression in CRCs is associated with poor clinical outcomes. Necroptosis can be engaged by common chemotherapeutics such as 5-fluorouracil (5-FU) to kill a subset of CRC cells with RIP3 expression, and is associated with a robust antitumor immune response. We also identified a novel necroptosis pathway involving the BH3-only Bcl-2 family protein PUMA, which activates RIP3 and MLKL to initiate necroptosis in response to anticancer agents. Based on these findings, we propose to test the hypothesis that PUMA/RIP3-mediated necroptosis plays a critical role in determining therapeutic response in a subset of CRC cells via both cell intrinsic and immunologic effects, which can be targeted to improve CRC therapy. Aim 1: Define the context and mechanism of necroptosis induction in CRC cells by anticancer agents; Aim 2: Delineate the functional role of necroptosis in the killing of CRC cells by anticancer agents; and Aim 3: Determine if manipulating necroptosis can be used to overcome therapeutic resistance of CRCs. The proposed studies will provide new mechanistic insights on necroptosis induction by anticancer agents in CRC cells. They will clarify how the interplay of apoptosis and necroptosis mediates response to anticancer therapy, and provide proof-of-principle evidence for stimulating necroptosis to enhance tumor cell killing and antitumor immune response for improving CRC treatment.